Human mitochondria maintain a small circular DNA that encodes several subunits of the proteins involved in oxidative phosphorylation as well as ribosomal and transport RNA. Defects in mtDNA expression result in a number of myopathies such as MELAS (mitochondrial encephalomyopath, lactic acidosis, and stroke-like syndrome), MERRF (myoclonic epilepsy with ragged red fibers syndrome), hearing and vision loss, and others. Moreover, mitochondria are implicated in age-related pathology, senescence and cancer and thus represent a rational but understudied therapeutic target. The key enzyme involved in mtDNA expression, mitochondrial RNA polymerase (mtRNAP) belongs to a family of single-subunit RNAPs that is distinct from the multi-subunit cellular RNAPs but distantly related to RNAPs of bacteriophage T7, the pol I familty of DNA polymerases, and single-subunit RNAPs from chloroplasts. However, unlike T7 RNAP, human mtRNAP requires two transcription factors, TFAM and TFB2M for efficient initiation, and its transcription appears to be regulated by a number of transcription factors. Studies of the structure and function of the mtRNAP and molecular mechanisms of its transcription are important for understanding the regulation of mitochondrial genome expression. This, in turn, will determine our ability to influence various mitochondrial functions and as a consequence, to treat mitochondria-associated diseases. The goal of this project is to determine the molecular mechanisms of transcription initiation by human mtRNAP. The specific aims are as follows: Aim 1. Determine the molecular basis of transcription initiation by mtRNAP. The high resolution structure of mtRNAP will be used to guide biochemical experiments to probe the function of mtRNAP domains that are involved in promoter binding, recognition, melting and interactions with transcription factors. Transitions of mtRNAP during initiation to elongation will be probed by cross-linking and by structural analysis of the mtRNAP elongation complex. Aim 2. Determine function and structure of the pre-initiation complex. Using protein-protein cross-linking we will determine the organization of a novel transcription intermediate - pre-initiation complex formed with TFAM and mtRNAP on promoter DNA. Interacting regions in both TFAM and mtRNAP will be mapped and their functional importance probed by mutagenesis. The structure of the pre-initiation complex will be determined at the atomic resolution and its function probed in various biochemical assays. Aim 3. Determine function and structure of the open promoter complex. Protein-protein cross-linking using artificial photo reactive amino acid will be used to test the model of TFB2M interaction with mtRNAP during initiation and to probe assembly of the initiation complex. The role of TFB2M in transcription start site selection, promoter specificity and promoter melting will be examined. The crystal structure of the core initiation complex that includes mtRNAP, TFAM, TFB2M and promoter DNA will be determined.